Department of Neuroscience and Neurology (K.P., T.G., H-M.M., J.J.), University of Kuopio, Kuopio, Finland
the Department of Neurology (J.S.), Kuopio University Hospital and Brain Research and Rehabilitation Center Neuron, Kuopio, Finland.
Abstract
Background and Purpose— The present study examined the long-term presence of -amyloid precursor protein (APP) and -amyloid (A) accumulation in the rat thalamus after focal cerebral ischemia.
Methods— Male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 2 hours. Sensorimotor outcome was assessed using a tapered/ledged beam-walking task after operation. The distribution of APP and A was examined immunohistochemically at 1 week, 1 month, and 9 months after MCAO.
Results— MCAO caused a long-lasting deficit in forelimb and hind limb function assessed using the beam-walking test. Histologic examination revealed a transient increase in APP and A staining in axons in the corpus callosum and in neurons at the border of the ischemic region. APP and A deposits persisted in the thalamic nuclei (ventroposterior lateral and ventroposterior medial nuclei), eventually leading to dense plaque-like deposits by the end of the 9-month follow-up. The deposits were surrounded by an astroglial scar. The deposits were positive for A and N-terminal APP, but not for C-terminal APP. Antibodies against the C-terminal of A, ie, A42 and A40, showed a preferential staining for A42. Congo red or thioflavine S did not stain the deposits.
Conclusions— The present results demonstrated the persistent presence and aggregation of APP and A, or their fragments, to dense plaque-like deposits in the ventroposterior lateral and ventroposterior medial nuclei of rats subjected to focal cerebral ischemia.
Key Words: amyloid cerebral ischemia rats thalamus
Introduction
Beta ()-amyloid precursor protein (APP) is a transmembrane protein with a long extracellular N-terminal and short intracellular C-terminal domain. APP is widely expressed in the brain, where its abnormal upregulation can lead to the accumulation of -amyloid (A), for example in Down syndrome patients.1,2,3 A is a hydrophobic self-aggregating peptide consisting of 40 to 42 residues, derived by sequential processing of the -amyloid precursor protein by -secretase and -secretase.4 A is a major component of senile plaques and is one of the pathologic hallmarks of Alzheimer disease.5
Brain trauma leads to the accumulation of a number of proteins, mostly as a consequence of the interruption of fast anterograde axonal transport.6 The accumulated proteins include APP and its proteolytic product A, neurofilament proteins, and synuclein proteins. Accumulated proteins usually disappear over time,7 but APP has been detected for up to 1 year after injury.8
Cerebral ischemia also leads to a transient upregulation and accumulation of APP.9,10 For example, APP staining and expression are detected in the subcortical white matter and adjacent to the boundary of the ischemic lesion in the gray matter after transient occlusion of the middle cerebral artery (MCAO).10–13 These studies, however, used only short survival periods ranging from days to weeks. The aim of the present study was to assess possible long-term accumulation of APP and A during a 9-month follow-up in rats subjected to transient MCAO.
Materials and Methods
Animals
Male Wistar rats (3 months old, 285 to 325 grams at the beginning of the study; National Laboratory Animal Centre, Kuopio, Finland) were used in the present study. The animals had free access to food and water and were housed in individual cages in a temperature-controlled environment (20±1°C) with lights on from 0700 to 1900 hours. Experimental procedures were conducted in accordance with the European Community Council directives 86/609/EEC and the study was approved by the Ethics Committee of the University of Kuopio and the Provincial Government of Kuopio.
Focal Cerebral Ischemia Model
Focal cerebral ischemia was induced using the intraluminal filament technique.14 Anesthesia was induced in a chamber using 3% halothane in 30% O2/70% N2O. A surgical depth of anesthesia was maintained throughout the operation with 0.5% to 1% halothane delivered through a nose mask. To occlude blood flow to the right MCA territory, a heparinized nylon filament ( 0.25 mm, rounded tip) was advanced 1.9 to 2.1 cm into the internal common carotid artery until resistance was felt. The filament was held in place by tightening the suture around the internal common carotid artery and placing a microvascular clip around the artery. Body temperature was monitored and maintained at 37°C using a heating pad connected to a rectal probe. After 120 minutes of MCAO, the filament was removed and the external carotid artery was permanently closed by electrocoagulation. Successful MCAO was verified on postoperative day 2 using the limb-placing test, which correlates with infarct volume.15
Beam-Walking Test
Forelimb and hind limb function were tested using a tapered/ledged beam (Figure 1).16 The animals were tested before and 1, 2, 4, 7, 12, 16, 20, and 32 weeks after surgery. The beam-walking apparatus consisted of a tapered beam with underhanging ledges on each side to permit foot faults without falling. The rats’ performance was videotaped and later analyzed by calculating the slip ratio (number of slips/number of total steps) of the impaired (contralateral to lesion) and nonimpaired (ipsilateral to lesion) forelimbs and hind limbs. Steps onto the ledge were scored as a full slip and a half-slip was scored if the limb touched the side of the beam. The mean of 3 trials was used for statistical analyses. All behavioral analyses were performed by an observer blind to the experimental groups.
Histology
Rats (MCAO, n=6; sham, n=7) were perfused transcardially 9 months after operation with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. Additional groups of rats were used for histologic purposes at 1 week (MCAO, n=10; sham, n=6) and 1 month (MCAO, n=10; sham, n=7) after operation. The brains were removed from the skulls, postfixed, and cryoprotected. Frozen sections (30 μm) were cut with a sliding microtome and stored in a cryoprotectant tissue collection solution at –20°C. One series of sections was stained with Toluidine Blue (Nissl stain) and the rest were processed for immunohistochemistry.17 Briefly, A was stained using a rodent-specific antibody (rabbit anti-rodent A3–16, #9151; Signet, Delham, Mass) or antibodies against the C-terminal end of A, ie, A42 (rabbit polyclonal, #9134; Signet) or A40 (rabbit polyclonal, #9131; Signet), or stained with FCA18 (A, specific for amino acid Asp-1; Dr Checler, France).18 APP was stained with 22C11 (mouse anti-APP, N-terminal; Chemicon, Temecula, Calif) or anti-APP C-terminal (rabbit anti-APP, C-terminal; Synaptic Systems, Gttingen, Germany). Glial fibrillary acidic protein (GFAP) was stained as a marker for astrocytes (mouse anti-GFAP; St. Louis, Mo) and OX-42 as a marker of microglia (mouse anti-rat CD11b; Serotec, Oxford, UK). The sections destined for APP and A staining were pretreated for 30 minutes with hot (85°C) citrate buffer. This series of sections was transferred to a solution containing the primary antibody (rabbit anti-rodent A at 1:5000; or A42 at 1:1000, A40 at 1:1000; FCA18 at 1:33 000; or 22C11 at 1:5000; or anti-APP at 1:20 000; or anti-GFAP at 1:1000; or anti-OX-42 at 1:4000) and Tris-buffered saline with 0.5% Triton X-100. After incubation in this solution for 18 hours on a shaker table at room temperature (20°C) in the dark, the sections were rinsed 3 times in Tris-buffered saline with 0.5% Triton X-100 and transferred to a solution containing the secondary antibody (goat anti-mouse*biotin; Sigma Chemical Co, St. Louis, Mo; or sheep anti-mouse Ig*biotin, Serotec). After 2 hours, the sections were rinsed 3 times with Tris-buffered saline with 0.5% Triton X-100 and transferred to a solution containing mouse ExtrAvidin (Sigma Chemical Co), and then incubated for 3 minutes with Ni-enhanced diaminobenzidine.17 A small number of sections were stained with Congo Red or thioflavine S.
Estimation of Infarct Volumes
Infarct volumes were measured using an image analysis system (MCID) from Nissl-stained sections collected at 1-mm intervals. The infarct area was determined according to the indirect method of Swanson et al19 by an observer blind to the experimental groups.
Statistics
Beam-walking data for the overall group effect were analyzed using analysis of variance for repeated measures.
Results
Histologic analysis revealed severe corticostriatal damage in all MCAO rats. The infarct volumes (mean±SD) were 81.6±23.1 mm3 in the cortex and 21.7±3.3 mm3 in the striatum. Analyses of beam-walking data revealed a significant difference in affected forelimb and hind limb function between MCAO and sham groups (F1,11=24.29; P<0.001 and F1,11=13.56, P<0.005, respectively). There was no significant group by day interaction in either forelimb or hind limb outcome. This indicates that MCAO induced an enduring deficit in sensorimotor function in beam-walking performance.
In MCAO rats that survived 1 week, there was an increase in APP staining (both N- and C-terminal) around the ischemic area, in the corpus callosum in crossing axons, in descending axons leaving the lesioned area, and in the terminal zone of these axons in the thalamus (Figure 2A1). Similarly, there was positive A staining in all of these areas (Figure 2A2). After 1-month survival, some N- and C-terminal APP staining was present around the ischemic area, but most APP was present at the terminal zone of the deafferentated axons in the thalamus (Figure 2B1). At 9 months after the MCAO, APP staining was not present around the lesioned area, in the corpus callosum, or in descending axons leaving the lesioned area (Figure 2C1). In the thalamus in the terminal zone of the deafferentated corticothalamic axons, however, there were large, dense deposits that resembled plaques and consisted of N-terminal APP (Figure 2F). These deposits were also positively stained for A, but not for C-terminal APP. The differential evolution of C-terminal and N-terminal APP in the cortex adjacent to the infarct area (penumbra), corticostriatal axons, and thalamus is shown in Figure 3. Relative staining intensities for antibodies against the C-terminal of A40 and A42, and A1-x, increased in that order (Figure 4B, 4C, and 4D). Congo red or thioflavine S did not stain, or only very lightly stained, the deposits (Figure 4A).
At 1 week after MCAO, the ischemic area was surrounded by activated astrocytes and microglia (data not shown). At 1 month, the number of activated glial cells had decreased substantially. At 9 months, there were no glial cells at the border of the ischemic region. In the ventroposterior lateral and ventroposterior medial nuclei (VPL/VPM), however, the time course of glial activity was quite different. At 1 week after MCAO, very few microglia and astrocytes were present (Figures 2G and 5A2). At 1 month, however, a large number of microglial cells and astrocytes were activated in the VPL/VPM (Figures 2H and 5B2). In contrast, at 9 months after MCAO, the dense A deposits were surrounded by an astroglial scar (Figure 5C2), but there were no activated microglia present (Figure 2I).
Discussion
In the present study, an increase in APP (both C-terminal and N-terminal) and A staining was observed in areas adjacent to the infarct, the corpus callosum, and the thalamus for 1 week after MCAO. The staining of these proteins later disappeared from the cortical areas and white matter but was still evident in the thalamus 9 months after MCAO. The N-terminal APP and A staining in the thalamus was diffuse acutely after the infarct, but accumulated, leading to dense plaque-like deposits in the VPL/VPM, a finding that has not been reported previously in naive or treated wild-type or transgenic rats.
APP is a marker of axonal injury and accumulates because of the disruption of fast anterograde axonal transport.6 After MCAO, APP immunoreactivity is acutely localized within axonal swellings, dystrophic neurites, and neuronal perikarya all along the periphery of the infarct, similar to the findings of our study. APP is present in reactive astrocytes in the periphery of the infarct from 3 to 60 days after MCAO.13 Astrocytes in the periphery are A immunoreactive from 7 to 30 days after ischemia, but this did not result in permanent A depositions, and there was no A immunoreactivity at 60 days. In the present study, there was a similar disappearance of APP and A staining in the ischemic border zone. An unexpected finding, however, was the transformation of the diffuse APP and A staining to dense plaque-like deposits in the VPL/VPM. It is unlikely that the accumulation of APP and A in the VPL/VPM was caused by indirect arterial changes caused by the intraluminal filament model, because similar changes are observed after fluid percussion injury6 and in other stroke models (unpublished observations) that do not involve arterial manipulation.
During the follow-up period, the APP and A staining pattern in the thalamus changed from diffuse staining to dense plaque-like deposits. In addition, although during the acute phase there was both C-terminal and N-terminal APP staining in the thalamus, likely in dying axons and at the synaptic terminals, the deposits that eventually formed were positive only for N-terminal APP, suggesting that the C-terminal APP was cleared over time, whereas the N-terminal APP slowly aggregated. More importantly, A antibodies used to further characterize the deposits showed a differential staining. First, there was a difference in staining intensities between C-terminal–specific antibodies, indicating preferential deposition of A42. This is consistent with a high A42/A40 ratio for rodent peptides compared with that for human peptides.20 The most intense staining, however, was with the A antibody, which recognizes A3–16, and for the antibody that recognizes A1-x. A possible explanation for this difference might be the presence of shorter peptides truncated at the carboxyl-terminus, such as A1 to 38, in the pool of soluble and insoluble A peptides.20 It has been suggested that cleavage at position 38 results in the formation of the A1 to 38 fragment because of a shift in -secretase activity.21 Whether the deposits are located intracellularly or extracellularly needs to be confirmed by electron microscopy, but our data with N-terminal APP and A antibodies indicated preferential extracellular location. Congo red or thioflavine S did not stain the deposits, but to our knowledge there is no evidence that rat A fragments form fibrils with -sheet conformation.22
Given that the accumulation of A is associated with neurodegeneration after brain injury,23 acute necrotic damage and a long-lasting degenerative process are likely to contribute to the present data. Based on the immunohistochemical data, it was difficult to differentiate whether acute axonal APP and A staining is related to degeneration of ascending thalamocortical fibers or descending corticothalamic fibers. The only neurons stained for APP and A, however, were present near the injury site. Thus, APP and A staining in the cortical border zone is most likely related to damaged corticothalamic neurons and axons. MCAO also leads to delayed secondary cell death in the thalamus outside the MCA arterial territory because of axonal damage of the thalamic neurons leading to retrograde degeneration.24 Vasogenic edema and excitatory noxious substances that spread with the edema fluid are additional pathogenic factors involved in the MCAO model.25 Hypometabolism and hypoperfusion typical to the intraluminal method might also have an effect on pathologic alterations in the thalamus.26,27 In addition, A produced by the brain is normally partially cleared by the interstitial fluid along the perivascular drainage pathways.28 Because thalamic nuclei, such as the VPL and VPM, are at the end artery area, it is possible that this clearance is insufficient.29 A is also eliminated by phagocytosis by microglia and astrocytes.30,31 A aggregations in the extracellular space were surrounded by microglia and astrocytes in the present study at the early time points (eg, 1 week and 1 month). At the latest age, only an astroglial scar was present, the formation of which might further impair the clearance of accumulated proteins.
In conclusion, MCAO caused transient accumulation of APP and A in the corpus callosum and ischemic border zone. APP and A staining was also evident in the thalamus where an early pattern of diffuse staining transformed to protein deposits in MCAO rats. These data might not be related to the initial sensorimotor impairment in MCAO rats, although plaque-like deposits in the thalamus are most likely harmful for functional recovery. Rather, the data might provide insight into possible mechanisms related to the pathologic protein accumulation commonly observed in neurodegenerative diseases. For example, Alzheimer disease is characterized by amyloid deposits and it has been suggested that progression of the pathology in Alzheimer disease is related to the connections between the areas displaying early deposits and the cortical regions showing later pathology.32 The study suggests that APP and A are transported through axons and secreted at terminals where they form diffuse deposits, which over time develop into plaque-like structures. Our data strongly support a similar process in MCAO rats, ie, axonal transport of APP and A fragments, extracellular deposition, and transformation to dense deposits. These findings might aid in furthering our understanding of the generation, deposition, and clearance of amyloid deposits in Alzheimer disease.
Acknowledgments
We thank Nanna Huuskonen and Pasi Miettinen for excellent technical assistance. We thank Dr Checler for the gift of the FCA18 antibody.
Footnotes
T.G. and K.P. contributed equally to this work.
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